Is There a Link Between Autism and Glyphosate-Formulated Herbicides?

Journal of Autism ISSN 2054-992X | Volume 3 | Article 1

Review Open Access Is there a link between autism and glyphosate-formulated herbicides?

James E. Beecham1 and Stephanie Seneff2*

*Correspondence: [email protected] CrossMark ← Click for updates

1Consultant Pathologist (Retired) University of Florida, Gainesville, USA. 2MIT Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, USA.

Abstract Glyphosate is the most widely used herbicide on the planet, and its increasing use over time in the United States aligns well with the increasing rates of autism determined by the Centers for Disease Control. Based on the known mechanism of glyphosate toxicity, we hypothesize that a pregnant woman’s exposure at mid- pregnancy to glyphosate-formulated herbicides (GFH) may produce, in her unborn child’s brain, anatomic alterations of cortical neuron layering remarkably similar to those found in the brains of humans with autism. Glyphosate’s known ability to chelate manganese ions combined with evidence of severely depleted serum manganese in cows exposed to glyphosate makes it likely that glyphosate would induce manganese deficiency in humans, interfering with the function of manganese-dependent enzymes. In particular, this would affect the maternal pituitary’s manganese-dependent Protein Phosphatase 1 (PP1) enzyme, resulting in a significant reduction in maternal serum levels of Thyroid-Stimulating Hormone (TSH). A study of mid-pregnancy maternal TSH serum levels in human mothers has found a statistical correlation of reduced TSH to increased risk of autism in offspring. Since insufficient thyroid stimulation by TSH or by iodine deficiency would both induce hypothyroidism, effects of iodine deficiency can be expected to emulate effects of TSH deficiency. Cortical neuron disarrangements have been produced in the brains of offspring of rat dams fed an iodine-deficient diet, and such foci of disordered cortical neurons are characteristically found in human autistic brains. While the research literature on glyphosate’s endocrine disrupting effects is limited, diverse evidence from animal studies reveals effects that suggest impaired thyroid function. If our hypothesis can be substantiated by a focused research effort, it would provide further justification for reducing or, ideally, eliminating glyphosate-formulated herbicide exposures in pregnant women. Keywords: Autism, glyphosate, manganese, hypothyroidism, topoisomerase, protein phosphatase, herbicide, neurodevelopment

Introduction that the only pesticide whose usage rates have gone up dra- The prevalence of autism has been rising at an alarming rate matically exactly in step with the rise in autism is glyphosate, in the United States over the past two decades, as determined and it is the most used herbicide on the planet. Pesticides are for example by the progressive rise in the number of children clearly toxic as that is their intent, and a strong correlation in in the public school system designated as autistic under the the rise of a serious disorder with a toxic chemical can not be Individuals with Disabilities Education act (IDEA). The pattern simply dismissed. of this rise is extremely well matched temporally to the rising An initiative undertaken by the organization, Moms Across use of glyphosate on corn and soy crops, as pointed out in America, has found levels of glyphosate in breast milk ranging an article by Swanson et al., and illustrated in their figure 23, from 76 mg/l to 166 mg/l in 3 out of 10 test samples (see: http:// reproduced here as Figure 1 [1]. The data plotted in this figure www.momsacrossamerica.com/ glyphosate testing results). are readily available from U.S. government sources on the Web, A European study of glyphosate residues in urine of humans and the correlation is nearly perfect (0.99) (p<1.1E-6). While and animals found significantly more glyphosate in the urine correlation does not necessarily mean causation, it is striking of humans eating a conventional diet compared to those eat-

© 2016 Seneff et al; licensee Herbert Publications Ltd. This is an Open Access article distributed under the terms of Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0). This permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Beecham et al. Journal of Autism 2016, http://www.hoajonline.com/journals/pdf/2054-992X-3-1.pdf doi: 10.7243/2054-992X-3-1

Autistic brains, during fetal development, appear to experience a disorder of neuron migration, at least in small foci. Stoner et al., found that autistic brains have small scattered foci of abnormal neocortex in which the usual 6 layers of neurons are instead blurred and ill-defined [4]. In addition to disorder in arrangement, some neurons in autism remained scattered within the white matter, apparently unable to migrate properly through the white matter and into the gray matter (Figure 3) as would normally occur. Neurons within these disordered foci in autistic brains also have a reduced content of calcium- buffering molecules, as demonstrated by reduced CALB1 in-situ hybridization staining. Lopez-Hurtado et al., found autistic brains to have a focal reduction in number of neurons and a focal increase in number of glial cells in areas related to speech processing [5].

Figure 1. Trends over time in the number of children with autism according to the IDEA and in the use of glyphosate on corn and soy crops. These data are readily available from the U.S. government from Web-based resources. Figure reproduced with permission from Swanson et al. [73]. ing an organic diet (P<0.0001) [2]. Furthermore, humans with chronic diseases had higher levels than those who were healthy. In this paper, we develop the hypothesis that glyphosate’s potential link to autism can be explained in part through an adverse effect on the thyroid gland of both the mother and the child during gestation. In the developing human brain, in order to form proper neuronal circuits and cortical architecture, neurons must migrate and assemble in an organized manner. Figure 3. Schematic of neocortex neuron arrangement, normal Under normal circumstances, immature neurons migrate (left) versus focus of abnormal cortex in autistic brain (right). through the paraventricular white matter to assemble in the Note the autistic brain has small foci with excess neurons, a neocortex and progressively form, within the gray matter, a blurring of layering, and numerous neurons still remaining distinct 6 layered pattern of neurons [3] (see Figure 2). within the white matter.

Figure 2. (Modified from Grieg et al. [3]) The 6 layered pattern of neocortical neurons. In humans, this pattern forms during in-utero fetal development during weeks 12 to 20. In the uppermost layer, the CajalRetzius cells (shown in orange) produce reelin, a protein which assists in properly arranging the migrating neurons. Definitions: VZ: Ventricular zone; SVZ: Subventricular zone; SP: Subplate.

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During neurodevelopment in children with autism, there is an Review excess of focal neurons in the early stages as compared to the Autism is a complex disorder of brain development charac- same age in typical fetal/childhood development, up to 70% terized by repetitive behavior patterns and difficulties with more neurons in foci early on in autistic brains as compared social communication and interaction. It is believed to usually to normal neurodevelopment. In the neocortex, these extra originate in utero, although there is also a form of regressive neurons migrate improperly, forming blurred indistinct layer- autism that occurs postnatally, which is not the subject of ing, with many neurons remaining within the white matter. this paper. Maternal hypothyroidism is linked to autism in Stoner et al., found such a pattern in 10 of the 11 autistics the fetus [9,10] and there is evidence from the neurological they studied, and also in one control brain, of a child who research literature supporting this link. We begin here with had suffered seizures [4]. Many of these excess disordered a review of selected features regarding the normal thyroid- neurons proceed through apoptosis at a later stage, and related physiology relevant to our theory. As is well-known, this programmed cellular death appears to stimulate local the in-utero fetus is heavily dependent upon the mother’s gliosis/glial activation. production of thyroid hormone for neurodevelopment (see In a previous article on autism [6], we presented evidence Figure 4). Maternal T4 (thyroxine) and T3 (triiodothyronine) supporting our theory that glyphosate may play a causal role cross the placenta and interact with cells in the fetus [11]. in autism through its mimicry of the amino acid glycine. We analyzed data suggesting that glyphosate’s mimicry would disrupt calcium inflow to immature neurons. For example, glyphosate may act at the glycine receptor of neurons (GlyR) which is a key to modulating the amount of chloride outflow from the immature neuron. This could cause inappropriate neuron membrane depolarizations, which then could force haphazard dislodgements of the magnesium ion from its blocking position within the N-methyl-D-aspartate (NMDA) receptor channel at the neuron membrane. This magnesium ion dislodgment could lead to excess and haphazard inflows of calcium into the immature neuron (if simultaneously the glycine binding site and glutamate binding site of the NMDA receptor are also agonistically bound). Glyphosate would plausibly induce excess bioavailability of glutamate by interfering with manganese-dependent conversion of glutamate to glutamine. One signal that such excess calcium inflows are happening to neurons in autistic brains would be the finding of a reduction in the calcium buffering capacity remaining in neurons. Stoner et al., demonstrated just such a reduced calcium buffer content within poorly migrating autistic neurons [4]. The inter- ested reader is referred to our previous paper for more details. Figure 4. (Modified from Bernal [45]) Maternal exposure to glyphosate theorized to decrease maternal Thyroid-Stimulating In [7], it was proposed that manganese chelation by Hormone (TSH), thus reducing T4 supply to fetus. glyphosate can explain a number of human pathologies that are currently on the rise, including autism. A study by Krüger et al., in 2013 showed that all the cows on 8 different Danish Fetus dependent on maternal thyroid hormone supply dairy farms that were fed genetically modified Roundup®- The active form of thyroid hormone, T3, is produced from T4 Ready corn and soy feed had extremely low serum levels of by the action of a deiodinase enzyme (D2), found for example manganese and cobalt [8]. The levels were well below the within astrocytes (see Figure 5). D2 removes an iodine from minimum of the typical range of values expected. In [7], it a molecule of T4 to produce T3, the active molecule. So criti- was discussed how low manganese bioavailability would cal is the proper level of T3 to the developing neuron that lead to impaired mitochondrial function due to disabled another deiodinase, D3, acts within the neuron to reduce manganese-dependent superoxide dismutase (Mn-SOD) as the T3 concentration, should it get too high. In the case of well as an impaired ability to detoxify glutamate by convert- maternal hypothyroxinemia, a reduced supply of maternal ing it to glutamine, using a manganese-dependent enzyme. T4 can be at least partially corrected by the astrocyte, such Both mitochondrial impairment and glutamate toxicity are as by increasing its D2 activity [10]. Increased D2 activity in linked to autism. Here, we focus on the effects of manganese the fetal astrocyte will more avidly de-iodinate the available deficiency on the disruption of phosphatase activity, and the T4, so as to compensate for the reduced T4 supply from ramifications leading to low thyroid activity. the mother. Another compensatory mechanism involves

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Figure 5. (Modifed from Bernal [45]) Activity of deiodinases within astrocytes and neurons. increased expression of thyroid hormone receptor genes, as we discuss below. Therefore, to evaluate whether glyphosate reduces T4 availability, we might look for increases in either D2 or beta thyroid hormone receptor mRNA, both of which may be Figure 6. (Modified from BMJ thyroid testing overview induced within a fetus facing maternal hypothyroxinemia online) Feedback system for thyroid hormones, Thyrotropin-Releasing Hormone (TRH), TSH, T3, T4. secondary to glyphosate dosing. In fact, glyphosate-formulated Glyphosate exposure of mother theorized to decrease herbicide exposure alters gene expression for thyroid hor- maternal TSH, thus reducing maternal T4/T3. mone receptor beta and deiodinase enzyme, with different alterations depending on stage/gender [12]. Specifically, in tadpoles, exposure to glyphosate-formulated herbicide is the TRH receptor, due to insufficient manganese to support linked to an increase in expression of thyroid receptor beta protein phosphatase 1 (PP1) activity, leading to reduced in brain [13], presumably as a defense mechanism in the face release of TSH, as explained below. of low T4. Furthermore, developmental delay was linked to glyphosate-formulated herbicide exposure in tadpoles [14]. Metal chelation by glyphosate/GFH In 1978, Madsen et al., in their report on the chemical proper- The pituitary/thyroid feedback control system ties of glyphosate, noted its propensity to form stable chelates The normal levels of TSH, T4 and T3 in the body are controlled with metal cations [16]. From kinetics characterized in figure 2 within a feedback system, as shown in Figure 6. The hypothala- in their paper, one can predict that, at the physiologic pH mus detects decreased T4 and/or decreased T3 levels in the range of approximately 7.4, in an idealized liquid medium blood. In response to low levels, the hypothalamus produces with adequate glyphosate, up to half of the manganese and releases thyrotropin-releasing hormone (TRH). Passing cations could be bound in stable chelate with glyphosate, through the hypophyseal portal channel to the pituitary, TRH thus reducing their bio-availability. At lower pH levels even binds with the TRH receptor of the thyrotrope in the anterior less manganese would be available. pituitary. The thyrotrope receptor for TRH is a G-protein coupled Mervosh et al. [17], writing in the journal Weed Science in receptor which binds TRH (see Figure 7). Following binding 1991, reported further on this well-known cation chelating of TRH, the receptor may then become displaced from the capacity of glyphosate, indicating that it appears to occur cell membrane, engulfed within a clathrin-coated vesicle and intracellularly, i.e., within plant cells, due to action of adju- processed inside the thyrotrope (10). vants promoting penetration of glyphosate into the cell. They In a clinical study of autistic boys, Hashimoto found a blunted proposed that cation chelation is the main mechanism by TSH response to TRH stimulation [15]. We believe this can be pre- which glyphosate exerts its lethal action in killing weeds. In dicted to be a consequence of manganese deficiency. Or- the years that followed, scientists began to explore the pos- dinarily, the process of TRH receptor binding of TRH causes sibility that glyphosate/GFH exposure in animals produced the release of TSH from the pituitary. The alpha TSH subunit harmful effects in a similar manner, including by glyphosate combines with the beta TSH subunit to form active TSH within chelating cations in food, thus reducing metal cation distribu- the thyrotrope (Figure 9). However, in the case of glyphosate tion/bioavailability, and/or by molecular action directly within exposure, there would be interference with the re-cycling of the body, including within animal cells [7,8,18-21].

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Figure 7. Thyrotrope of pituitary, normal TSH production.

Studies have documented that glyphosate also chelates zinc well-known ability to chelate divalent metal cations [22,23], [16,22,23]. Because zinc is required for the synthesis of thy- we hypothesize that glyphosate gains entry within the thyroid hormones, it is possible that lack of bioavailable zinc is rotrope and chelates ionic manganese, interfering with PP1 a contributory factor in hypothyroidism [24]. Supplementing dephorphorylation (see Figure 8). zinc could conceivably lessen the chance of hypothyroidism. As schematically illustrated in Figure 8, where the co-factor As supportive evidence, Tizhe et al., documented a protec- manganese is chelated by glyphosate, and PP1 is thus unable tive effect of zinc supplementation in Wistar rats dosed with to de-phosphorylate the receptor, the result is reduced TSH glyphosate [25]. release. Multiple animal studies on glyphosate have provided evidence that pituitary cells in animals exposed to glyphosate Details of the thyrotrope, TRH receptor function, PP1 exhibit increased protein content of beta TSH subunits (i.e., enzyme the beta subunits accumulate un-assembled) [29,30]. As shown in Figure 7, the normal signal processing of TRH within the pituitary involves a recycling of the TRH recep- Impaired TSH production tor, from the membrane to the cell interior and back to the TSH formation typically occurs from the joining together membrane, which depends upon the action of PP1. of an inactive alpha TSH subunit and an inactive beta TSH The TRH receptor, after binding with TRH, is typically lo- subunit. When these are successfully joined (Figure 9), the TSH cated within a vesicle inside the thyrotrope. PP1 removes the can be exported to the circulation in active form. The alpha bound phosphate from the receptor, by dephosphorlyating TSH subunit is nearly identical to that of human chorionic the receptor/TRH combination. This permits the assembly gonadotropin (hCG), luteinizing hormone (LH) and follicle- and release of TSH from the pituitary [26]. Typically, the TRH stimulating hormone (FSH). The TSH beta subunit confers receptor, after de-phosphorylation, is enabled to move back the specificity to TSH. to re-join the cell membrane, where it can become available GFH exposure causes the beta TSH subunits to accumulate to bind another molecule of TRH. unused within the thyrotrope cytoplasm. A study by Romano Importantly, as demonstrated in studies by Gerhet and et al., presents findings of glyphosate dosing of pregnant rats Hinkle [27,28], manganese is a necessary co-factor for PP1’s which includes data of male offspring pituitaries having the dephosphorylation of the receptor. Due to glyphosate’s predicted beta TSH subunit accumulation [29]. In addition, male

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Figure 8. Thyrotrope with manganese chelated by glyphosate, resulting in inactivation of PP1. TSH release is thus reduced.

thyroid stimulation due to exposure to GFH. Lavado-Autric et al., studied the effect of iodine deficiency on fetal neuron migration [31]. They examined the brains of the offspring of pregnant rat dams fed an iodine deficient diet, and found autism-like neuron migratory defects in the fetal/neonate brains. Others have also linked iodine deficiency and autism. Hamza et al., studied 50 autistic Egyptian children and their mothers compared to 50 control pairs [32]. They found that iodine deficiency was prevalent in both the children and their mothers, with a statistically significant inverse relationship Figure 9. TSH is assembled from an alpha subunit and a beta between iodine content and disease severity. They speculated subunit. A particular segment of the beta subunit, known as the buckle or seatbelt, wraps around the alpha subunit to form that iodine deficiency could cause autism. the active hormone. This seatbelt region also helps stabilize Glyphosate-induced hypothyroidism might produce signs the hormone’s structure. and symptoms in an animal (or in a human) beyond those caused by iodine deficiency. For example, various symptoms and signs within various organs and systems may reflect GFH’s offsprings’ pituitary content of beta TSH mRNA was reduced [30], manganese-chelation inactivation of PP1. PP1 influences presumably due to product inhibition of the unassembled glycogen’s mobilization into glucose when muscle cells re- beta subunit. The defect, we believe, is at the level of the quire glucose [33]. If such activity is reduced by glyphosate’s PP1 enzyme, putatively due to lack of manganese co-factor. chelating of manganese within the body, the animal’s muscles may function poorly, producing muscle/activity-related signs. Iodine deficiency versus protein phosphatase deficiency Slower weight gain might also be expected due to the poor Iodine deficiency is an important factor in hypothyroidism, mobilization of glycogen during animal growth. A study on and the effects are likely more acute in the context of impaired glyphosate exposure to dogs found that the female dogs ex-

6 Beecham et al. Journal of Autism 2016, http://www.hoajonline.com/journals/pdf/2054-992X-3-1.pdf doi: 10.7243/2054-992X-3-1 posed to 926mg/kg bw per day of glyphosate showed a gradual to 70% of their normal level. reduction in growth rate over time, compared to controls, Importantly, the rat dams did not demonstrate clinical which was consistently significant from week 23 onwards [34]. signs of hypothyroidism. Yet, when the male offspring were Okadaic acid is a toxin from shellfish which is known to inhibit sacrificed at day 40 postnatal and their brains examined, 83% protein phosphatases. Okadaic acid overdose often presents demonstrated significant alteration of neuronal arrangement with diarrhea [35]. Diarrhea is much less likely to be seen in in the cortex with numerous misplaced neurons. Furthermore, iodine deficiency-induced hypothyroidism. A study by Koch there were functional alterations in the behavior of the et al., found that phosphatase inhibitors okadaic acid and offspring rats. When the offspring were exposed at day 39 calyculin-A both inhibited corticotropin-releasing factor postnatal to acoustic stimuli, 53% of them responded with (CRF)-induced secretion of ACTH in AtT-20 corticotropes [36]. wild runs, which were followed in some offspring by seizures. A regulatory study exposing rats to glyphosate found that Reduced maternal serum TSH level in mid-pregnancy has soft stools and/or diarrhea occurred in both genders at all been linked to increased risk of autism in human offspring doses [37]. This could be an effect of inhibition of protein [9,10]. While not easily observed or detected in animal studies, phosphatase, similar to that of okadaic acid overdose. Several research has demonstrated that, in humans, maternal hypo- manganese-dependent prokaryotic protein phosphatases thyroxinemia during pregnancy appears linked to a reduction with numerous biological roles have been identifed [38], in the offspring child’s IQ, as well as to impaired attention, and therefore decreased manganese bioavailability can be language, and visual-motor performance at 7-9 years [44-46]. expected to disrupt the gut microbiome in unpredictable ways. Altered thyroid hormone receptor expression in the brain Impaired selenoprotein function Under normal circumstances, thyroid hormone receptors Gut commensals provide many essential nutrients to the expressed in the fetal brain are predominantly of the alpha body [39]. In particular, Lactobacillus participates in bacte- 1 type. T3 binds to these fetal brain receptors in order to acti- rial fixation of inorganic selenium into more bioavailable vate genes in the nucleus [47]. By contrast, thyroid hormone organic forms such as selenocysteine and selenomethionine. receptor beta (THR beta) usually has only low expression in Lactobacillus critically depends on manganese for protection the fetal period, and in normal circumstances demonstrates against oxidative damage [40,41]. Lactobacillus reduction due an increased expression only during the postnatal period and to glyphosate’s chelation of manganese [18] might therefore through adulthood. However, with pregnancy complicated by reflect reduced selenoprotein availability. Iodothyroninedeio- maternal hypothyroxinemia, compensatory mechanisms in dinases, glutathione peroxidase and thioredoxin reductase the brain of the in utero fetus are likely to include an increase are all selenoproteins, and these enzymes are involved in in the fetal brain’s expression of thyroid hormone receptor thyroid hormone metabolism, regulation of redox state and beta [45,48,49]. protection from oxidative damage [42]. In tadpoles, glyphosate-formulated herbicides alter the expression of key genes involved in development, with Fetal neuron migration due to maternal hypothyrox- changes in mRNA levels differently affected depending on inemia, litter size/weight developmental stage [13]. There was a clear dose-response As mentioned above, in a study of rat dams fed an iodine effect for a glyphosate-formulated herbicide to increase deficient diet [31], researchers sacrificed male offspring at thyroid hormone receptor beta expression in tadpole brain 40 days postnatal and examined the brains. The fetal cortex [13]. Tadpoles also have shown increased time to metamor- of the sacrificed male rats displayed misplaced neurons and phosis [14]. Delay in growth/development of earthworms was a blurring of the cortical layering. demonstrated with glyphosate exposure in environmentally The researchers also noted that feeding the rat dams an relevant amounts [50]. iodine-deficient diet had not reduced litter size or litter total weight, or offspring body weight, or offspring postnatal Myxedema coma and acute glyphosate poisoning growth measures. Hence, we should acknowledge that neuron Severe hypothyroidism can lead to myxedema coma, par- migration defects can occur in an animal without also finding ticularly among the elderly under conditions of stress disruption of litter size, litter weight, offspring body weights, such as acute infection [51]. It is associated with low body or other changes to postnatal growth measures. temperature, hypoventilation and respiratory acidosis, low A key factor to consider is how short the maternal expo- blood pressure, hyponatremia, hypercapnia, hypoxia, and sure to a factor disruptive of thyroid function can be to still slowed and irregular heart rate. Many of these symptoms are induce neuron migration defects in offspring. In a study by characteristic features of acute glyphosate poisoning due to Auso et al., researchers used rat dams and applied only a 3 attempted suicide [52,53]. For example, hypercapnia, respira- day course of a goitrogen during the period of early fetal tory distress necessitating intubation, altered consciousness, neurodevelopment [43]. The goitrogen was demonstrated low blood pressure, hypoxemia and shock are all listed as to have depressed maternal thyroid hormones transiently acute reactions to glyphosate poisoning. Hypercapnia leads

7 Beecham et al. Journal of Autism 2016, http://www.hoajonline.com/journals/pdf/2054-992X-3-1.pdf doi: 10.7243/2054-992X-3-1 to respiratory acidosis, which is also a characteristic feature and a significant elevation of IL-6 (394 mg/ml). All signs and of acute glyphosate poisoning. symptoms suggesting meningitis resolved as the concentra- A case study of an elderly woman who was admitted to tion of glyphosate in CSF decreased. She was discharged after hospital in a state of coma is illustrative of the dramatic effect 39 days of hospitalization. that can occur through induction of hypothyroidism through Mice exposed to glyphosate-rich air or glyphosate alone in poisoning [54]. The woman had consumed an overabundance air developed high eosinophil and neutrophil counts, mast cell of uncooked bok choy, which possesses an enzyme capable degranulation, and production of IL-33, thymic stromal lym- of blocking thyroid function. Emergency treatment included phopoietin (TSLP), and IL-13 [63]. TSLP induces IL-6 expression thyroid hormone injections and she eventually recovered. and is linked to asthma, chronic obstructive pulmonary disease, However, untreated myxedema coma comes with a very high and atopic dermatitis [64]. IL-6 promotes a chronic inflamma- 50% mortality rate. tory response by stimulating T-cells and B-cells [65]. This is In reviewing data from animal studies of glyphosate/GFH significant because inflammation is known to be capable of dosing, we should be watching for similar thyroid-related damaging neurodevelopment [66,67]. Increased thymus weight collapse in animals. Death following glyphosate exposure in male mice during glyphosate exposure at intermediate and may be a consequence of endocrine collapse. Hypothermia highest doses hints at harmful glyphosate action via inflam- was noted in a regulatory study of rats exposed to glyphosate, matory thymus hyperplasia, most prominent in males [34]. along with subdued behavior with hunched posture and decreased activity 6 hours after dosing in 3 of 10 females at Other manganese-dependent enzymes the highest dose [37]. This is suggestive of endocrine reduc- Numerous enzymes within the human body depend on tion/protein phosphatase inactivation symptoms. One female manganese as a co-factor. Thus, removing bioactivity of man- of the 3 was found dead on day 2 of the study. Researchers ganese by chelation could allow a toxin to cause symptoms originally ascribed this death to general toxicity of glyphosate, in several organ systems, simultaneously [7]. For example, but in view of the possibility of myxedema coma, deaths manganese is essential for mitochondrial protection from are suggestive of endocrine failure. Another female animal, oxidative damage, the production of energy from glucose, from the lowest dose group, also died, again approximately the growth of bones, the development of the skeleton and 6 hours after dosing on day 1. The short half-life of TSH [55] the formation of cartilage, among other key functions [68,69]. may render glyphosate capable of inducing endocrine col- Therefore, in reviewing animal studies, we should watch for lapse in just a few hours. any signs related to these multiple functions. While excess manganese in the body is known to be toxic, Inflammation and cytokines a deficiency of manganese has been found to produce mal- As suggested in a study by Hsiao et al., in circumstances development and mal-functioning of nerves and muscles where inflammation in the mother passes cytokines to the [70,71]. Multiple studies have linked autism to manganese placenta/fetus, there might be a reduction in the overall deficiency. One study showed reduced manganese content total weight of a litter [56]. They found that cytokines can in hair and/or urine as compared to controls [72], and another inhibit growth hormone activity. Furthermore, Wajner et study showed reduced manganese levels in the enamel of al., noted that IL-6-induced oxidative stress reduces deio- deciduous teeth [73]. Mn-SOD is essential for protecting the dinases D1 and D2, thus reducing conversion of T4 to T3, mitochondria from oxidative damage, and it requires man- as well as increasing D3-mediated inactivation of T3 [57]. ganese as a co-factor. In a study of autistic children under 6 Dahlgren et al., found that IL-6 passes the placental barrier years of age, SOD activity (presumably including Mn-SOD [57]. Wei et al., noted that IL-6 is active in the brain [59,60] activity) was found to be significantly reduced as compared and they theorized that IL-6 causes adverse action in neu- with age-matched controls [74]. rodevelopmental diseases, including mediating autism- Estimates suggest that in nearly one-third of enzyme- like signs and symptoms. In a separate report, Wei et al., mediated processes, metal ion co-factors are required for found elevated IL-6 in the cerebellum of autistic brains [60]. activity. For example, topoisomerase (TI) enzymes use divalent Glyphosate has been shown to induce an inflammatory metal cations for their activity, principally magnesium and response in both acutely-exposed humans and in in animal manganese [75]. TI enzymes are key for proper gene expres- studies. Sato et al., reported the case of a 58 year old woman sion, especially with regard to long kilobase genes. There who had ingested approximately 150 ml of a glyphosate- are a number of different types of topoisomerases, each formulated liquid (41% glyphosate and 15% polyoxethyl- specializing in a different aspect of DNA manipulation such eneamine) [62]. Two days later she was admitted to hospital as unwinding/breaking-reattaching DNA strands (TP1) so that in acute respiratory distress with circulatory collapse, renal DNA can be copied/replicated, DNA supercoiling adjustments, failure, disseminated intravascular coagulopathy and aseptic and (TP2-gyrase) alternately condensing/de-condensing DNA. meningitis. A sample of her cerebrospinal fluid was tested King et al. [76] recently reported that many of the genes and demonstrated the presence of glyphosate (122.5 mg/ml) regulated by topoisomerases are involved in the function of

8 Beecham et al. Journal of Autism 2016, http://www.hoajonline.com/journals/pdf/2054-992X-3-1.pdf doi: 10.7243/2054-992X-3-1 synapses, the junctions between neurons, and many of these PP2A might be similarly inactivated by GFH exposure. The IARC genes are also linked to autism. King et al., cross-referenced report mentioned that PP2A, under ordinary circumstances, their list of genes regulated by topoisomerases with autism “maintains cell homeostasis by counteracting most of the candidate genes catalogued by various sequencing studies kinase-driven intracellular signaling pathways.” Indeed, the and by SFARI Gene, a comprehensive database of genes report called PP2A “a bona fide tumor suppressor.” linked to autism. They found that 49 of the 183 candidate autism genes were affected by topoisomerases –27 percent Discussion –a proportion much higher than would be seen by chance. This paper has developed the hypothesis that glyphosate In our view, TI enzyme inhibition via manganese chelation may be contributing to the autism epidemic in part through could explain the high incidence of discordant sibling muta- its suppression of thyroid hormone production. We have tions in autism cases, as found for example by Yuen et al., in presented supporting evidence from a diverse literature, whole genome sequencing of quartet families [77]. In other including epidemiological evidence from temporal correla- words, via the same manganese-chelation mechanism, one tions, regulatory and other animal toxicology studies, studies child in a family might suffer a disabling/mutation of one par- characterizing the symptoms of acute glyphosate poisoning, ticular autism-related gene, while his/her sibling might suffer and the research literature relating autism in the offspring to a disabling/mutation of a different autism-related gene. One maternal hypothyroidism. possibility as to the level at which these disparate effects might In order to clarify explicitly whether autism-type changes occur would be at the level of paternal spermatozoa [78-80]. can be caused in fetal brains due to maternal exposure to glyphosate/GFH from consumption of common food and Autism, hypothyroidism and mid-pregnancy TSH beverages, relevant animal dosing studies need to be care- Hypothyroidism in Western countries is often associated fully designed. Such studies are increasingly important to with Hashimoto’s thyroiditis, an autoimmune condition. As fund and conduct because the incidence of autism is rising mentioned above, in non-Western areas of the world, iodine alarmingly. If exposure to glyphosate/GFH from common deficiency is the predominant cause of hypothyroidism. With food and beverages is a factor in autism, governmental and iodine deficiency, hypothyroid symptoms and low serum medical authorities need such information, as do prospective T4 are typically found in the face of elevated serum TSH. By parents, in particular pregnant women. contrast, we would expect glyphosate/GFH dosing in animals, via manganese chelation, to cause hypothyroidism with a Considerations for study design reduced serum TSH level. Autism in laboratory animals has a significant social interaction Such a reduction in serum TSH was found in a study and behavioral component. Observers carrying out standard of pregnant women in California with regard to autism animal-testing protocols for regulatory chemical toxicity spectrum disorder (ASD) risk in offspring. Yau et al. studied assessment are likely unaware of the appropriate signs of maternal mid-pregnancy serum samples and infant blood autistic behaviors, such as alterations in scent marking or samples after birth in regard to three groups of children vocalization distress. Furthermore, they are not specifically born in Orange County, CA in 2000-2001 including ASD looking for autism-like behaviors in their assessment of toxic- (n=78), developmental delay (n=45), and general popula- ity effects. Autism is associated with changes originating at tion controls (GP) (n=149) [10]. Adjusted logistic regression the early stages of fetal brain development. Therefore, the models showed inverse association between ASD and nature of toxic effects must be addressed in the context of log transformed mid-pregnancy TSH levels in maternal the pregnant animal and in utero fetus, and carefully evalu- serum samples (ASD vs. GP: OR [95% CI] 0.33 0.12-0.91]). ated by appropriate protocol. This is not within the standard design typically used in chemical toxicity testing in animals PP2A and cancer for regulatory purposes. Guyton et al., recently reported, in Lancet Oncology, the One can gain statistical significance by considering the decision of the International Agency for Research on Cancer entire litter. In a 2013 study, Swiss researchers reviewed sev- Monograph Working Group (IARC) to designate glyphosate eral earlier studies of autism using the valproic-acid-injected as a ‘probable carcinogen [81]. Ironically, two years earlier, animal model [84]. They concluded that 91% (31 out of the 34 a report appeared in that same journal [82] indicating that studies) suffered from statistical limitations, due to a failure protein phosphatase 2A (PP2A) had been found genetically to use the litter as a level of statistical analysis, rather than altered or functionally inactivated in many solid cancers just analyzing the individual mouse or rat. This will be an and leukemia. It is known that PP2A requires manganese as important consideration in the design of future experiments co-factor [83]. on glyphosate. While the animal dosing studies we will review do not In humans, the critical period during which fetal neurons focus on the issue of cancer, if glyphosate/GFH exposure in migrate into the neocortex is approximately between the humans is capable of inactivating PP1, it is conceivable that 12th through 20th weeks of gestation [85]. By comparison, the

9 Beecham et al. Journal of Autism 2016, http://www.hoajonline.com/journals/pdf/2054-992X-3-1.pdf doi: 10.7243/2054-992X-3-1 critical period in rats tends to be from embryonic day E12 to duced the normal increase of mRNA for Thyroid Receptor beta E19 of gestation [86]. Thus, studies of GFH dosing in animals during developmental Gosner stages Gs37-42 [12]. However, should include a carefully planned histologic examination of at Gs42, only males demonstrated this effect, suggesting that the brain according to a specific and detailed protocol designed females are hormonally protected in utero. This is a possible to expose pregnant dams during the critical period and detect explanation of male predominance in autism incidence. neuron migration defects in the offspring. Anything less will not be able to assess the types of changes within the cortical Conclusions layering pattern of neurons which appear to be key to finding Many different factors have been purported to be linked to autism-type alterations. Also, gene expression and enzyme autism. These have included, among others, genetic factors, activities related to neurodevelopment should be assessed. The iodine deficiency, vitamin D deficiency, and toxic exposures standard chemical toxicity testing protocols obviously do not to a variety of substances, including lead, mercury, aluminum, include such evaluations. Nevertheless, we believe that there thimerosal, fluoride, particulate matter in air pollution, to can be value in reinterpreting the data from earlier toxicol- name a few. Whether, and to what magnitude, glyphosate/ ogy studies in light of new insight gained from knowledge of GFH have a place on the list is still debatable. plausible mechanisms, provided we understand the limitations. This paper presents a hypothesis that glyphosate exposure In regard to glyphosate/GFH, and based on our review of in utero can cause developmental defects leading to autism evidence from the scientific literature, a number of associated due in part to impaired thyroid function in the mother and in research areas also appear likely to yield meaningful informa- the fetus. Hypothyroxinemia in rat dams fed an iodine-deficient tion. Several of these are listed below, with a brief summary of diet (15) caused neuron migration defects in the brains of evidence already found in various studies on glyphosate, along offspring reminescent of those observed in association with with relevant citations from the published scientific literature. autism. If glyphosate causes even borderline hypothyroxinemia in mothers exposed to glyphosate-formulated herbicides, as Glyphosate/GFH and the disabling of pituitary protein studies we suggest be carried out might definitively establish, phosphatase 1 (PP1) then we see no reason to believe such exposure could not Perinatal exposure to glyphosate in rat dams decreased the produce, in human fetuses, similar disordered neuron migration. content of beta subunit TSH mRNA in male rat offspring pi- Glyphosate can be expected to impair thyroid function both tuitary, while increasing pituitary beta subunit TSH protein through depletion of manganese essential for the production content [29]. A related study in humans [10] showed that of TSH in the pituitary and through inhibition of the synthesis maternal TSH level mid-pregnancy was found to be inversely of multiple selenoproteins involved in thyroid function. proportional to the risk of ASD in offspring. Other features Compelling evidence comes from research by Yau et al., in presented studies which suggest hypothyroidism from which showed that a decrease in mid-pregnancy maternal glyphosate exposure are subdued behavior [33], hypothermia TSH correlates statistically with a higher risk of ASD in the [63], decreased physical activity [34,37], hunched posture offspring of such pregnancies [10]. Although only trace [36] and decreased growth rates/body weights, especially amounts of glyphosate/GFH are thought to be present in in females [34]. commonly consumed food and beverages, even such small exposures at critical times in development might cause Glyphosate/GFH and endocrine collapse autism-type neuron migration defects in the in-utero fetus. The symptoms of acute glyphosate poisoning include several Glyphosate-resistant weeds growing among genetically that are specifically associated with myxedema coma due engineered Roundup® -Ready crops have necessitated ever to excess stress under hypothyroidism [52,53]. Decreased increasing use of glyphosate on core crops of the processed survival of tadpoles from glyphosate-formulated exposure food industry. Furthermore, the now widespread practice of was apparently endocrine related [13]. In various rat stud- desiccation, i.e., the spraying of crops with glyphosate just ies, possible endocrine failure was suggested by several days before the harvest, on wheat, sugar cane, legumes, and unexplained animal deaths following glyphosate dosing, other crops, has further raised residue levels in staple crops especially in females [34]. and therefore in many processed foods. These foods are not Soft stools/diarrhea in rats and muscle/skeletal dysfunction, required to be labelled with the level of GFH residue present, as suggested by hunched posture [34], suggest other aspects and are almost never tested. of PP1 inactivation. In tadpoles exposed to glyphosate for- We have reviewed in a broader context evidence of thyroid mulations containing POEA, decreased snout-vent length at dysfunction in association with glyphosate exposure, and metamorphosis and increased time to metamorphosis, tail have found support related to acute glyphosate poisoning damage, and gonadal abnormalities were found [14]. and myxedema coma in humans, as well as impaired growth, lowered IQ, hypothermia, and decreased physical activity, all Glyphosate/GFH and toxic action by gender related to hypothyroidism. In tadpoles, glyphosate-formulated herbicide exposure re- We urgently encourage researchers with appropriate

10 Beecham et al. Journal of Autism 2016, http://www.hoajonline.com/journals/pdf/2054-992X-3-1.pdf doi: 10.7243/2054-992X-3-1 resources to consider carrying out carefully designed stud- References ies, including glyphosate/GFH dosing to pregnant animals 1. Swanson N L, Leu A, Abrahamson J and Wallet B. Genetically engineered while using glyphosate-free feeding diets for control animals. crops, glyphosate and the deterioration of health in the United States of America. Journal of Organic Systems. 2014; 9. Examination of offspring should include detecting signs of 2. Krüger M, Schledorn P, Schrödl H-W, Lutz W and Shehata AA. Detection autistic behavior in animals. Also, careful histologic review of glyphosate residues in animals and humans. J Environ Anal Toxicol. of brain tissue should be carried out to specifically look for 2014; 4:2. signs of fetal neuron migration defects and/or other features 3. Greig LC, Woodworth MB, Galazo MJ, Padmanabhan H and Macklis JD. commonly found in autistic brains. Molecular logic of neocortical projection neuron specification, development and diversity. Nat Rev Neurosci. 2013; 14:755-69. | Article | We acknowledge that we have not yet proven that glypho- PubMed Abstract | PubMed FullText sate/GFH exposure from common food and beverages causes 4. Stoner R, Chow ML, Boyle MP, Sunkin SM, Mouton PR, Roy S, Wynshaw- human autism. On the other hand, we have perhaps high- Boris A, Colamarino SA, Lein ES and Courchesne E. Patches of disorga- lighted glyphosate’s/GFH’s molecular potential, through a nization in the neocortex of children with autism. N Engl J Med. 2014; 370:1209-19. | Article | PubMed Abstract | PubMed FullText set of deduced mechanisms. This theory will have served its 5. Lopez-Hurtado E and Prieto J J. A Microscopic Study of Language-Relat- purpose if it spurs appropriate future research, including as ed Cortex in Autism Am. J Biochem Biotech. 2008; 4:130-45. | Pdf outlined above. If that research confirms our hypothesis, it will 6. Beecham J E and Seneff S. The possible link between Autism and also have enabled strategies to effectively reduce the incidence Glyphosate Acting as Glycine Mimetic – a review of evidence from the literature with analysis. J Molec Genet Med. 2015; 9:4. | Article of autism. Regardless, mothers are advised to consume and 7. Samsel A and Seneff S. Glyphosate, pathways to modern diseases III: feed their family an organic diet as a precautionary measure. Manganese, neurological diseases, and associated pathologies. Surg Neurol Int. 2015; 6:45. | Article | PubMed Abstract | PubMed FullText List of abbreviations 8. Krüger M, Schrödl W, Neuhaus J and Shehata A A. Field Investigations of ACTH: Adrenocorticotropic hormone Glyphosate in Urine of Danish Dairy Cows. J Environ Anal Toxicol. 2013; CALB1: Calbindin 1 3:186. | Article CRF: Corticotrophin-releasing factor 9. Roman GC, Ghassabian A, Bongers-Schokking JJ, Jaddoe VW, Hofman A, GFH: Glyphosate-formulated herbicide de Rijke YB, Verhulst FC and Tiemeier H. Association of gestational -ma GlyR: Glycine receptor of neurons ternal hypothyroxinemia and increased autism risk. Ann Neurol. 2013; hCG: Human chorionic gonadotropin 74:733-42. | Article | PubMed IARC: International Agency for Research on Cancer 10. Yau VM, Lutsky M, Yoshida CK, Lasley B, Kharrazi M, Windham G, Gee N LH: Luteinizing hormone and Croen LA. Prenatal and neonatal thyroid stimulating hormone levels and autism spectrum disorders. J Autism Dev Disord. 2015; 45:719-30. | MnSOD: Mitochondrial superoxide dismutas Article | PubMed NMDA: N-methyl- D-aspartate receptor 11. Sack J. Thyroid function in pregnancy - maternal-fetal relationship in POEA: Polyethoxylatedtallowamine health and disease. Pediatr Endocrinol Rev. 2003; 1 Suppl 2:170-6. | PP1: Protein phosphatase 1 Article | PubMed THR: Thyroid hormone receptor 12. Lanct ̈ot C, Navarro-Martín L, Robertson C, Park B, Jackman P, Pauli B D TRH: Thyrotropin-releasing hormone and Trudeau V L. Effects of glyphosate-based herbicides on survival, de- TSH: Thyroid- Stimulating Hormone velopment, growth and sex ratios of wood frog (Lithobates sylvaticus) TSLP: Thymic stromal lymphopoietin tadpoles. II: agriculturally relevant exposures to Roundup® Weath- erMax® and VisionMaxR®under laboratory conditions. Aquat Toxicol. Competing interests 2014; 154:291-303. The authors declare that they have no competing interests. 13. Navarro-Martin L, Lanctot C, Jackman P, Park BJ, Doe K, Pauli BD and Trudeau VL. Effects of glyphosate-based herbicides on survival, develop- Authors’ contributions ment, growth and sex ratios of wood frogs (Lithobates sylvaticus) tad- Authors’ contributions JEB SS poles. I: chronic laboratory exposures to VisionMax(R). Aquat Toxicol. 2014; 154:278-90. | Article | PubMed Research concept and design ✓ ✓ 14. Howe CM, Berrill M, Pauli BD, Helbing CC, Werry K and Veldhoen N. Collection and/or assembly of data ✓ ✓ Toxicity of glyphosate-based pesticides to four North American frog species. Environ Toxicol Chem. 2004; 23:1928-38. | Article | PubMed Data analysis and interpretation ✓ ✓ 15. Hashimoto T, Aihara R, Tayama M, Miyazaki M, Shirakawa Y and Kuroda Y. Writing the article ✓ ✓ Reduced thyroid-stimulating hormone response to thyrotropin-releasing hormone in autistic boys. Dev Med Child Neurol. 1991; 33:313-9. | Critical revision of the article ✓ ✓ Article | PubMed Final approval of article ✓ ✓ 16. Madsen H E and Christensen H H. Gottleib Petersen C Stability Constants Statistical analysis ✓ ✓ of Copper (II), Zinc, Manganese (II), Calcium and M agnesium Com- plexes of N-(Phosphonomethyl) glycine (Glyphosate) Acta Chemica Acknowledgement and funding Scandanavica A. 1978; 32:79-83. This research is supported in part by Quanta Computers, 17. Mervosh T L and Balke N E. Effects of Calcium, Magnesium and Phos- Taiwan, under the auspices of the Qmulus project. phate on Glyphosate Absorption by Cultured Plant Cells. Weed Science. 1991; 39:347-53. | Article Publication history Editor: David Reiss, Imperial College London, UK. 18. Samsel A and Seneff S. Glyphosate, pathways to modern diseases II: Received: 06-Nov-2015 Final Revised: 09-Dec-2015 Celiac sprue and gluten intolerance. Interdiscip Toxicol. 2013; 6:159-84. | Article | PubMed Abstract | PubMed FullText Accepted: 11-Jan-2016 Published: 18-Jan-2016

11 Beecham et al. Journal of Autism 2016, http://www.hoajonline.com/journals/pdf/2054-992X-3-1.pdf doi: 10.7243/2054-992X-3-1

19. Krüger M, Schledorn P, Schrödl W, Hoppe HW and Lutz W et al. Detection 37. Pfeil R, Niemann and L. Glyphosate. Federal Institute for Risk Assess- of Glyphosate Residues in Animals and Humans. J Environ Anal Toxicol. ment, Berlin, Germany. JMPR. 2004; 95-169. 2014; 4:210. 38. Shi L. Manganese-dependent protein O-phosphatases in prokaryotes 20. Séralini G, Clair E, Mesnage R and Gress S et al. Republished study: long- and their biological functions. Front Biosci. 2004; 9:1382-97. | PubMed term toxicity of a Roundup herbicide and a Roundup-tolerant geneti- 39. Morowitz MJ, Carlisle EM and Alverdy JC. Contributions of intestinal cally modified maize. Environmental Sciences Europe. 2014; 26:14. | Pdf bacteria to nutrition and metabolism in the critically ill. Surg Clin North 21. Gasnier C, Dumont C, Benachour N, Clair E, Chagnon MC and Seralini GE. Am. 2011; 91:771-85. | Article | PubMed Abstract | PubMed FullText Glyphosate-based herbicides are toxic and endocrine disruptors in hu- 40. Nierop Groot MN and de Bont JA. Involvement of manganese in man cell lines. Toxicology. 2009; 262:184-91. | Article | PubMed conversion of phenylalanine to benzaldehyde by lactic acid bacteria. 22. Jayasumana C, Gunatilake S and Senanayake P. Glyphosate, hard water Appl Environ Microbiol. 1999; 65:5590-3. | Article | PubMed Abstract | and nephrotoxic metals: are they the culprits behind the epidemic of PubMed FullText chronic kidney disease of unknown etiology in Sri Lanka? Int J Environ 41. Archibald FS and Duong MN. Manganese acquisition by Lactobacillus Res Public Health. 2014; 11:2125-47. | Article | PubMed Abstract | plantarum. J Bacteriol. 1984; 158:1-8. | Article | PubMed Abstract | PubMed FullText PubMed FullText 23. Caetano M S, Ramalho T C, Botrel D F, da Cunha E F and de Mello W 42. Schomburg L. Selenium, selenoproteins and the thyroid gland: interac- C. Understanding the Inactivation Process of Organophosphorus tions in health and disease. Nat Rev Endocrinol. 2012; 8:160-71. | Article Herbicides: A DFT Study of Glyphosate Metallic Complexes with Zn2, | PubMed Ca2, Mg2, Cu2, Co3, Fe3, Cr3, and Al3. International Journal of Quantum Chemistry. 2012; 112:27522762. | Article 43. Auso E, Lavado-Autric R, Cuevas E, Del Rey FE, Morreale De Escobar G and Berbel P. A moderate and transient deficiency of maternal thyroid 24. Betsy A, Binitha M and Sarita S. Zinc deficiency associated with hypothy- function at the beginning of fetal neocorticogenesis alters neuronal roidism: an overlooked cause of severe alopecia. Int J Trichology. 2013; migration. Endocrinology. 2004; 145:4037-47. | Article | PubMed 5:40-2. | Article | PubMed Abstract | PubMed FullText 44. Zimmermann MB, Aeberli I, Melse-Boonstra A, Grimci L, Bridson J, Cha- 25. Tizhe EV, Ibrahim ND, Fatihu MY, Onyebuchi, II, George BD, Ambali SF and ouki N, Mbhenyane X and Jooste PL. Iodine treatment in children with Shallangwa JM. Influence of zinc supplementation on histopathological subclinical hypothyroidism due to chronic iodine deficiency decreases changes in the stomach, liver, kidney, brain, pancreas and spleen during thyrotropin and C-peptide concentrations and improves the lipid pro- subchronic exposure of Wistar rats to glyphosate. Comp Clin Path. 2014; file. Thyroid. 2009; 19:1099-104. | Article | PubMed 23:1535-1543. | Article | PubMed Abstract | PubMed FullText 45. Bernal J. Thyroid hormone receptors in brain development and 26. Jiang X, Dias, J A and He X. Structural biology of glycoprotein hormones function. Nat Clin Pract Endocrinol Metab. 2007; 3:249-59. | Article | and their receptors: Insights to signaling. Molecular and Cellular Endocri- PubMed nology. 2014; 382:424451. | Article 46. Qian M, Wang D, Watkins WE, Gebski V, Yan YQ, Li M and Chen ZP. The 27. Gehret A U and Hinkle P M. Protein Phosphatase 1alpha is involved in effects of iodine on intelligence in children: a meta-analysis of studies the regulation of the thyrotropin-releasing hormone receptor. FASEB J. conducted in China. Asia Pac J Clin Nutr. 2005; 14:32-42. | Pdf | PubMed 2012; 26:lb180. | Article 47. Yamamoto N, Li QL, Mita S, Morisawa S and Inoue A. Inhibition of thy- 28. Hinkle PM, Gehret AU and Jones BW. Desensitization, trafficking, and roid hormone binding to the nuclear receptor by mobilization of free resensitization of the pituitary thyrotropin-releasing hormone recep- fatty acids. Horm Metab Res. 2001; 33:131-7. | Article | PubMed tor. Front Neurosci. 2012; 6:180. | Article | PubMed Abstract | PubMed FullText 48. Patel J, Landers K, Li H, Mortimer RH and Richard K. Thyroid hormones and fetal neurological development. J Endocrinol. 2011; 209:1-8. | 29. R. Romano, P. Souza, M. Nunes and M. Romano. Perinatal exposure to Article | PubMed a commercial formulation of glyphosate reduces the mRNA expression and increases the protein content of beta TSH in the pituitary of male 49. Dong H, You SH, Williams A, Wade MG, Yauk CL and Thomas Zoeller R. offspring. Endocrine Abstracts. 2012; 29:753. | Article Transient Maternal Hypothyroxinemia Potentiates the Transcriptional Response to Exogenous Thyroid Hormone in the Fetal Cerebral Cortex 30. Romano R, Souza P, Nunes M and Romano M. Perinatal exposure to a Before the Onset of Fetal Thyroid Function: A Messenger and MicroRNA commercial formulation of glyphosate reduces the mRNA expression Profiling Study. Cereb Cortex. 2015; 25:1735-45. | Article | PubMed and increases the protein content of beta TSH in the pituitary of male offspring. Endocrine Abstracts. 2012; 29:P753. 50. Santadino M, Coviella C and Moma F. Glyphosate Sublethal Effects on the Population Dynamics of the Earthworm Eisenia Fetida (Savigny 31. Lavado-Autric R, Auso E, Garcia-Velasco JV, Arufe Mdel C, Escobar del 1826). Water Air Soil Pollution. 2014; 225:2207. | Article Rey F, Berbel P and Morreale de Escobar G. Early maternal hypothyroxinemia alters histogenesis and cerebral cortex cytoarchitecture of the 51. Wall CR. Myxedema coma: diagnosis and treatment. Am Fam Physician. progeny. J Clin Invest. 2003; 111:1073-82. | Article | PubMed Abstract | 2000; 62:2485-90. | Article | PubMed PubMed FullText 52. Lee HL, Chen KW, Chi CH, Huang JJ and Tsai LM. Clinical presentations 32. Hamza RT, Hewedi DH and Sallam MT. Iodine deficiency in Egyptian -au and prognostic factors of a glyphosate-surfactant herbicide intoxica- tistic children and their mothers: relation to disease severity. Arch Med tion: a review of 131 cases. Acad Emerg Med. 2000; 7:906-10. | Article Res. 2013; 44:555-61. | Article | PubMed | PubMed 33. Brady MJ and Saltiel AR. The role of protein phosphatase-1 in insulin 53. Talbot AR, Shiaw MH, Huang JS, Yang SF, Goo TS, Wang SH, Chen CL and action. Recent Prog Horm Res. 2001; 56:157-73. | Pdf | PubMed Sanford TR. Acute poisoning with a glyphosate-surfactant herbicide (‘Roundup’): a review of 93 cases. Hum Exp Toxicol. 1991; 10:1-8. | 34. World Health Organization and Food and Agriculture Organization of the Article | PubMed United Nations. Pesticide residues in food – 2004. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and 54. Chu M and Seltzer TF. Myxedema coma induced by ingestion of raw bok the Environment and the WHO Core Assessment Group on Pesticide choy. N Engl J Med. 2010; 362:1945-6. | Article | PubMed Residues Rome, Italy, 2029 September 2004. 55. Szkudlinski MW, Fremont V, Ronin C and Weintraub BD. Thyroid-stimulat- 35. Yasumoto T, Murata M, Oshima Y, Sano M and Matsumoto G K et al. Diar- ing hormone and thyroid-stimulating hormone receptor structure-func- rhetic shellfish toxins. Tetrahedron. 1985; 41:1019-25. tion relationships. Physiol Rev. 2002; 82:473-502. | Article | PubMed 36. Koch B and Lutz-Bucher B. Inhibition of protein phosphatases by okadaic 56. Hsiao EY and Patterson PH. Activation of the maternal immune system acid and calyculin-A differentially modulates hormonal- and forskolin- induces endocrine changes in the placenta via IL-6. Brain Behav Immun. stimulated formation of cyclic AMP in AtT-20 corticotrophs: effect of 2011; 25:604-15. | Article | PubMed Abstract | PubMed FullText pituitary adenylate activating polypeptide and corticotropin-releasing 57. Wajner SM, Goemann IM, Bueno AL, Larsen PR and Maia AL. IL-6 pro- factor. Cell Signal. 1994; 6:467-73. | Article | PubMed motes nonthyroidal illness syndrome by blocking thyroxine activation

12 Beecham et al. Journal of Autism 2016, http://www.hoajonline.com/journals/pdf/2054-992X-3-1.pdf doi: 10.7243/2054-992X-3-1

while promoting thyroid hormone inactivation in human cells. J Clin In- 76. King IF, Yandava CN, Mabb AM, Hsiao JS, Huang HS, Pearson BL, Cal- vest. 2011; 121:1834-45. | Article | PubMed Abstract | PubMed FullText abrese JM, Starmer J, Parker JS, Magnuson T, Chamberlain SJ, Philpot 58. Dahlgren J, Samuelsson AM, Jansson T and Holmang A. Interleukin-6 in BD and Zylka MJ. Topoisomerases facilitate transcription of long genes the maternal circulation reaches the rat fetus in mid-gestation. Pediatr linked to autism. Nature. 2013; 501:58-62. | Article | PubMed Abstract | Res. 2006; 60:147-51. | Article | PubMed PubMed FullText 59. Wei H, Chadman KK, McCloskey DP, Sheikh AM, Malik M, Brown WT and 77. Yuen RK, Thiruvahindrapuram B, Merico D, Walker S, Tammimies K, Ho- Li X. Brain IL-6 elevation causes neuronal circuitry imbalances and medi- ang N, Chrysler C, Nalpathamkalam T, Pellecchia G, Liu Y, Gazzellone MJ, ates autism-like behaviors. Biochim Biophys Acta. 2012; 1822:831-42. | D’Abate L, Deneault E and Howe JL et al. Whole-genome sequencing of Article | PubMed quartet families with autism spectrum disorder. Nat Med. 2015; 21:185- 91. | Article | PubMed 60. Wei H, Alberts I and Li X. Brain IL-6 and autism. Neuroscience. 2013; 252:320-5. | Article | PubMed 78. Kong A, Frigge ML, Masson G, Besenbacher S, Sulem P, Magnusson G, Gudjonsson SA, Sigurdsson A, Jonasdottir A, Wong WS, Sigurdsson G, 61. Wei H, Zou H, Sheikh AM, Malik M, Dobkin C, Brown WT and Li X. IL-6 is Walters GB, Steinberg S, Helgason H, Thorleifsson G, Gudbjartsson DF, increased in the cerebellum of autistic brain and alters neural cell adhe- Helgason A, Magnusson OT, Thorsteinsdottir U and Stefansson K. Rate of sion, migration and synaptic formation. J Neuroinflammation. 2011; de novo mutations and the importance of father’s age to disease risk. 8:52. | Article | PubMed Abstract | PubMed FullText Nature. 2012; 488:471-5. | Article | PubMed Abstract | PubMed FullText 62. Sato C, Kamijo Y, Yoshimura K and Ide T. Aseptic meningitis in associa- 79. Degrassi F, Fiore M and Palitti F. Chromosomal aberrations and genomic tion with glyphosate-surfactant herbicide poisoning. Clin Toxicol (Phila). instability induced by topoisomerase-targeted antitumour drugs. Curr 2011; 49:118-20. | Article | PubMed Med Chem Anticancer Agents. 2004; 4:317-25. | Article | PubMed 63. Kumar S, Khodoun M, Kettleson EM, McKnight C, Reponen T, Grinsh- 80. Tamburrino L, Marchiani S, Montoya M, Elia Marino F, Natali I, Cambi M, pun SA and Adhikari A. Glyphosate-rich air samples induce IL-33, TSLP Forti G, Baldi E and Muratori M. Mechanisms and clinical correlates of and generate IL-13 dependent airway inflammation. Toxicology. 2014; sperm DNA damage. Asian J Androl. 2012; 14:24-31. | Article | PubMed 325:42-51. | Article | PubMed Abstract | PubMed FullText Abstract | PubMed FullText 64. Shan L, Redhu NS, Saleh A, Halayko AJ, Chakir J and Gounni AS. Thymic 81. Guyton KZ, Loomis D, Grosse Y, El Ghissassi F, Benbrahim-Tallaa L, Guha stromal lymphopoietin receptor-mediated IL-6 and CC/CXC che- N, Scoccianti C, Mattock H and Straif K. Carcinogenicity of tetrachlorvin- mokines expression in human airway smooth muscle cells: role of phos, parathion, malathion, diazinon, and glyphosate. Lancet Oncol. MAPKs (ERK1/2, p38, and JNK) and STAT3 pathways. J Immunol. 2010; 2015; 16:490-1. | Article | PubMed 184:7134-43. | Article | PubMed 82. Perrotti D and Neviani P. Protein phosphatase 2A: a target for anticancer 65. Gabay C. Interleukin-6 and chronic inflammation. Arthritis Res Ther. therapy. Lancet Oncol. 2013; 14:e229-38. | Article | PubMed Abstract | 2006; 8 Suppl 2:S3. | Article | PubMed Abstract | PubMed FullText PubMed FullText 66. Miller BJ, Culpepper N, Rapaport MH and Buckley P. Prenatal inflam- 83. Xing Y, Xu Y, Chen Y, Jeffrey PD, Chao Y, Lin Z, Li Z, Strack S, Stock JB and mation and neurodevelopment in schizophrenia: a review of human Shi Y. Structure of protein phosphatase 2A core enzyme bound to studies. Prog Neuropsychopharmacol Biol Psychiatry. 2013; 42:92-100. | tumor-inducing toxins. Cell. 2006; 127:341-53. | Article | PubMed Article | PubMed 84. Lazic SE and Essioux L. Improving basic and translational science by 67. Le Belle JE, Sperry J, Ngo A, Ghochani Y, Laks DR, Lopez-Aranda M, accounting for litter-to-litter variation in animal models. BMC Neurosci. Silva AJ and Kornblum HI. Maternal inflammation contributes to brain 2013; 14:37. | Article | PubMed Abstract | PubMed FullText overgrowth and autism-associated behaviors through altered redox signaling in stem and progenitor cells. Stem Cell Reports. 2014; 3:725-34. 85. Rabinowicz T, de Courten-Myers GM, Petetot JM, Xi G and de los Reyes | Article | PubMed Abstract | PubMed FullText E. Human cortex development: estimates of neuronal numbers indicate major loss late during gestation. J Neuropathol Exp Neurol. 1996; 68. Baly DL, Keen CL and Hurley LS. Pyruvate carboxylase and phosphoenol- 55:320-8. | Article | PubMed pyruvate carboxykinase activity in developing rats: effect of manganese deficiency. J Nutr. 1985; 115:872-9. | Article | PubMed 86. Ignacio MP, Kimm EJ, Kageyama GH, Yu J and Robertson RT. Postnatal migration of neurons and formation of laminae in rat cerebral cortex. 69. Armstrong CG, Browne GJ, Cohen P and Cohen PT. PPP1R6, a novel Anat Embryol (Berl). 1995; 191:89-100. | Article | PubMed member of the family of glycogen-targetting subunits of protein phosphatase 1. FEBS Lett. 1997; 418:210-4. | Article | PubMed 70. Claus Henn B, Ettinger AS, Schwartz J, Tellez-Rojo MM, Lamadrid-Figueroa H, Hernandez-Avila M, Schnaas L, Amarasiriwardena C, Bellinger DC, Hu Citation: H and Wright RO. Early postnatal blood manganese levels and children’s Beecham JE and Seneff S. Is there a link between neurodevelopment. Epidemiology. 2010; 21:433-9. | Article | PubMed autism and glyphosate-formulated herbicides? J Abstract | PubMed FullText 71. Zota AR, Ettinger AS, Bouchard M, Amarasiriwardena CJ, Schwartz J, Hu Autism. 2016; 3:1. H and Wright RO. Maternal blood manganese levels and infant birth http://dx.doi.org/10.7243/2054-992X-3-1 weight. Epidemiology. 2009; 20:367-73. | Article | PubMed Abstract | PubMed FullText 72. Akyuzlu D K, Kayaalti Z, Soylemez E and Soylemezoglu T. Association between Autism and Arsenic, Lead, Cadmium, Manganese Levels in Hair and Urine. J of Pharmacy and Pharmacol. 2014; 2:140-44. 73. Abdullah MM, Ly AR, Goldberg WA, Clarke-Stewart KA, Dudgeon JV, Mull CG, Chan TJ, Kent EE, Mason AZ and Ericson JE. Heavy metal in children’s tooth enamel: related to autism and disruptive behaviors? J Autism Dev Disord. 2012; 42:929-36. | Article | PubMed 74. Meguid NA, Dardir AA, Abdel-Raouf ER and Hashish A. Evaluation of oxidative stress in autism: defective antioxidant enzymes and increased lipid peroxidation. Biol Trace Elem Res. 2011; 143:58-65. | Article | PubMed 75. Sissi C and Palumbo M. Effects of magnesium and related divalent metal ions in topoisomerase structure and function. Nucleic Acids Res. 2009; 37:702-11. | Article | PubMed Abstract | PubMed FullText 13